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Microbacterium Awajiense Sp. Nov., Microbacterium Fluvii Sp. Nov. and Microbacterium Pygmaeum Sp. Nov

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Microbacterium Awajiense Sp. Nov., Microbacterium Fluvii Sp. Nov. and Microbacterium Pygmaeum Sp. Nov Actinomycetologica (2008) 22:1–5 Copyright Ó 2008 The Society for Actinomycetes Japan VOL. 22, NO. 1 Microbacterium awajiense sp. nov., Microbacterium fluvii sp. nov. and Microbacterium pygmaeum sp. nov. Akiko Kageyama1, Yoshihide Matsuo2, Hiroaki Kasai2, Yoshikazu Shizuri2, Satoshi O¯ mura1;3, and Yoko Takahashi1Ã 1Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan. 2Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan. 3The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan. (Received Jan. 15, 2008 / Accepted Feb. 25, 2008 / Published May 16, 2008) The taxonomic positions of three novel strains isolated from soil, driftwood and sediment samples collected in Japan were investigated based on the results of chemotaxonomic, phenotypic and genotypic character- istics. The strains that we examined were Gram-positive, catalase-positive bacteria with L-ornithine as a diagnostic diamino acid of peptidoglycan. The acyl type of peptidoglycan was N-glycolyl. The major menaquinones were MK-11, -12, -13 and/or -14. Mycolic acids were not detected. The G+C content of the DNA was 68 to 70 mol%. These morphological and chemotaxonomical characters and comparative 16S rDNA analysis of the three isolated strains revealed that they belong to the genus Microbacterium. DNA- DNA relatedness data revealed that the three isolates are three new species of the genus Microbacterium. On the basis of the polyphasic evidence, the isolates should be classified as novel species of the genus Microbacterium: Microbacterium awajiense sp. nov., Microbacterium fluvii sp. nov. and Microbacterium pygmaeum sp. nov. with the type strains YM13-414T (=MBIC08276T, DSM 18907T), YSL3-15T (=MBIC08277T, DSM 18908T) and KV-490T (=NRRL B-24469T, NBRC 101800T), respectively. INTRODUCTION EGG medium at 25 C for 30 days. The components of EGG medium are shown in Table 1. The bacterium was The genus Microbacterium was first proposed by then cultured on Marine Broth 2216 (Difco) containing Orla-Jensen (1919) with the type species Microbacterium 1.5% agar after being cultivated for 7–10 days. Strain lacticum, and was emended by Takeuchi & Hatano (1998). YSL3-15T was isolated from driftwood collected in The genus Microbacterium is a member of the family October 2005 at the estuary of Maera River on Iriomote Microbacteriaceae in the order Actinomycetales. In the Island, Japan. The driftwood was crushed in autoclaved present study, strain YM13-414T was isolated from a sedi- artificial seawater. Next, 1/10 diluents of the sample was ment sample, strain YSL3-15T was isolated from driftwood, applied to seawater medium containing 0.1% lignan. and KV-490T was isolated from a soil sample. On the pres- Colonies were isolated after incubation for 1 week at ent phenotypic and chemotaxonomic data strongly indicate 25 C. Strain KV-490T was isolated from a soil sample that these strains belong to the genus Microbacterium. Their collected in the Aoyama Cemetery in Tokyo, Japan. Two phenotypic and phylogenetic characteristics, coupled with grams of soil was suspended in 18 mL of sterile water and data for genomic DNA-DNA relatedness levels, suggest mixed. Soil particles were allowed to sediment, the liquid that these strains should be classified as the novel species phase was diluted to 105 and 100 mL samples were spread Microbacterium awajiense sp. nov., Microbacterium fluvii onto the surface of each plate. GPM agar plates (1.0% sp. nov. and Microbacterium pygmaeum sp. nov. glucose, 0.5% peptone, 0.5% meat extract, 0.3% NaCl and 1.2% agar, pH 7.0) with SOD (300 unit/plate) and catalase MATERIALS AND METHODS (2100 unit/plate) (Takahashi et al., 2003) were used, and these were cultured at 27 C. KV-490T was isolated from Bacterial strains and isolation GPM agar plates with SOD and catalase. Biomass for Strain YM13-414T was isolated from a sediment biochemical and chemotaxonomic characteristics was sample collected from the shore of Yura, Awaji Island, prepared by culturing in TSB broth at 27 C. Japan (depth 20 cm, GPS location: N 3416025.700,E 13457013.800), in September 2004. The samples (0.5– Morphological and biochemical tests 1cm3) were homogenized with a glass rod in 5 mL of Morphological observation under a scanning electron sterile seawater. The homogenate (50 mL) was cultured on microscope (model JSM-5600; JEOL) was performed using ÃCorresponding author and address: Yoko Takahashi, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan., phone: +81-3-5791-6133, e-mail: [email protected], fax: +81-3-5791-6133. 1 ACTINOMYCETOLOGICA VOL. 22, NO. 1 Table 1. Components of EGG medium. EGG medium ÃMetal mix X Metal mix X 250 mLÃ NaCl 500 g ÃÃ ‘‘EGG’’ mix 100 mL MgSO4Á7H2O 180 g Cycloheximide 50 mg CaCl2Á2H2O 2.8 g Gryseofluvin 25 mg KCl 14 g Nalidixic acid 20 mg Na2HPO4Á12H2O5g Aztreonam 40 mg FeSO4Á7H2O 200 mg Agar 20 g PII metals 600 mLÃÃÃ DW 650 mL/pH 7.6 S2 metals 100 mLÃÃÃÃ DW 4300 mL/pH 7.6 ÃÃEGG mix ÃÃÃPII metals Extract Ehlrich 0.5 g Na2-EDTA 1 g Hurt Infusion Broth 0.2 g H3BO3 1.13 g Gelatin 0.2 g Fe soln. 1 mL of FeCl3Á6H2O (2.42 g/50 mL) Glycerol 0.2 g Mn soln. 1 mL of MnCl2Á4H2O (7.2 g/50 mL) Yeast extract 0.2 g Zn soln. 1 mL of ZnCl2 (0.52 g/50 mL (+HCl)) DW 100 mL/pH 7.6 Co soln. 1 mL of CoCl2Á6H2O (0.2 g/50 mL) DW 996 mL/pH 7.5 ÃÃÃÃS2 metals NaBr 1.28 g Mo soln 10 mL of Na2MoO4Á2H2O (0.63 g/50 mL) Sr soln. 10 mL of SrCl2Á6H2O (3.04 g/50 mL) Rb soln. 10 mL of RbCl (141.5 mg/50 mL) Li soln. 10 mL of LiCl (0.61 g/50 mL) I soln. 10 mL of KI (6.55 mg/50 mL) V soln. 10 mL of V2O5 (1.785 mg/50 mL (+NaOH)) DW 940 mL/pH 7.5 cultures grown on GPM agar medium at 27 C for 3 or 6 equipped with a CAPCELL PAK C18 column (Shiseido days. The carbon-assimilation properties of the two strains Co., Ltd.) (Tamaoka et al., 1983). Methyl esters of cellular YM13-414T and KV-490T were determined using Pridham- fatty acids were prepared, and were analyzed by GLC Gottlieb agar medium (Nihon Pharmaceutical Co., Ltd.) (model HP6890; Hewlett-Packard). (Pridham & Gottlieb, 1948), and the carbon-assimilation properties of strain YSL3-15T were determined using ten- G+C content of DNA and DNA-DNA hybridization times diluted Pridham-Gottlieb agar medium supplemented DNA was isolated as described by Saito & Miura with ten-times diluted nutrient agar medium (Difco). NaCl (1963). DNA base composition was determined by HPLC tolerance and the pH and temperature ranges for growth (Tamaoka & Komagata, 1984). Levels of DNA-DNA were determined using 1/5 nutrient agar. The three isolates relatedness were assayed using the method of Ezaki et al. were characterized biochemically using API ZYM (bio- (1989) using photobiotin and a microplates format. Me´rieux) according to the manufacturer’s instructions. 16S rDNA sequencing and phylogenetic analysis Chemotaxonomic tests DNA was prepared by sonication (Yu et al., 2002) or The N-acyl type of muramic acid was determined using using InstaGene matrix (Bio-Rad). 16S rDNA was ampli- the method of Uchida & Aida (1977). Purified cell walls fied by PCR and sequenced with an automatic sequence were obtained using the method of Kawamoto et al. (1981). analyzer (ABI PrismÔ 3130 or 3730; PE Applied Bio- One milligram of purified cell walls was hydrolyzed at systems) using a dye terminator cycle sequencing kit (PE 100 C with 1 mL of 6 N HCl for 16 h. The residue was Applied Biosystems). dissolved in 100 mL of water, and was used for amino acid Species related to the new isolate were identified by analysis by thin layer chromatography (TLC). Mycolic acid performing sequence database searches using the BLAST was assayed using the TLC method of Tomiyasu (1982). program (Altsuchul et al., 1990). Sequence data of related Menaquinones were extracted and purified using the species were retrieved from GenBank. Nucleotide substi- method of Collins et al. (1977), and were then analyzed tution rates (Knuc values) were calculated (Kimura & Ohta, by HPLC (model 802-SC; Jasco) on a chromatograph 1972) and phylogenetic trees were constructed using the 2 ACTINOMYCETOLOGICA VOL. 22, NO. 1 Table 2. Fatty acid composition (%) of isolated strains. A B C YM13-414T YSL3-15T KV-490T iso-C15:0 14.04 3.48 1.18 anteiso-C15:0 31.69 48.43 30.45 iso-C16:0 16.65 18.06 7.17 C16:0 1.62 5.91 1.77 iso-C17:0 5.74 1.52 1.82 anteiso-C17:0 28.81 19.58 56.81 C18:0 — 1.48 — Fig. 1. Scanning electron micrographs of cells from 3- or 6-day- old cultures of strains YM13-414T (A), YSL3-15T (B) and KV- 490T (C) grown on GPM agar medium at 27 C. Bar, 2 mm. Microbacterium fluvii YSL3-15T (AB286028) 41 Microbacterium awajiense YM13-414T (AB286027) Microbacterium deminutum KV-483T (AB234026) 93 Microbacterium pumilum KV-488T (AB234027) Microbacterium flavum YM18-098T (AB286029) Microbacterium lacticum IFO 14135T (AB007415) 45 42 Microbacterium schleiferi DSM20489T (Y17237) Microbacterium koreense JS53-2T (AY962574) Microbacterium pygmaeum KV-490T (AB248875) Microbacterium terregens IFO 12961T (AB004721) T 68 Microbacterium lacus A5E-52 (AB286030) Microbacterium aurum DSM 8600T (Y17229) 64 Microbacterium aoyamense KV-492T (AB234028) Microbacterium terricolae KV-448T (AB234025) M. liquefaciens DSM 20638T (X77444) 0.002 Knuc Fig.
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